Human amniotic fluid: a source of stem cells for possible therapeutic use.

Stem cells are undifferentiated cells with the capacity for differentiation. Amniotic fluid cells have emerged only recently as a possible source of stem cells for clinical purposes. There are no ethical or sampling constraints for the use of amniocentesis as a standard clinical procedure for obtaining an abundant supply of amniotic fluid cells. Amniotic fluid cells of human origin proliferate rapidly and are multipotent with the potential for expansion in vitro to multiple cell lines. Tissue engineering technologies that use amniotic fluid cells are being explored. Amniotic fluid cells may be of clinical benefit for fetal therapies, degenerative disease, and regenerative medicine applications. We present a comprehensive review of the evolution of human amniotic fluid cells as a possible modality for therapeutic use.

Amniotic fluid as a source of multipotent cells for clinical use.

Amniotic fluid cells (AFC) from 2nd trimester amniocentesis have been found to be a source of multipotent stem cells which might overcome the limitations of expansion, histocompatibility, tumorigenesis, and ethical issues associated with using human embryonic cells, umbilical cord, cord blood, bone marrow, and induced pluripotent cells. Previous work by our group and others demonstrated multipotency and the ability to grow well in culture. However, all these studies were done in media containing fetal calf serum. We sought to observe the properties of AFC grown in serum-free media as that would be required for clinical transplantation in humans. Fresh samples were obtained from three patients, and each sample divided into a culture whose cells were not exposed to fetal calf serum, and the other half into a standard culture medium containing fetal calf serum. Doubling time and stem cell marker expression by flow cytometry were assessed. Differentiation to neural, osteoid, and chondrogenic lineages was induced using appropriate media and confirmed by fluorescent microscopy, histology, and immunohistochemistry. There were no statistically significant differences between cells grown serum-free and in standard media in any of these parameters. The data supports the possibility of clinical use of AFC in stem cell transplantation.

Identification and isolation of putative stem cells from the murine placenta.

OBJECTIVE: The placenta of mid-gestation mice is a known rich source of hematopoietic stem cells. We hypothesized that it is also a source of other multipotent stem cells. METHODS: We isolated fetal cells from the murine placenta across the second half of gestation and characterized their expression of surface antigens known to be associated with mesenchymal stem cells (MSCs) on a subset of hematopoietic lineage-negative cells. Using real-time reverse-transcriptase quantitative polymerase chain reaction, we also evaluated the expression of intracellular transcription factors (TFs) known to be associated with renal development and/or multipotent stem cells. RESULTS: Cell phenotypes with surface marker and TF expression consistent with multipotent stem cells of a mesenchymal lineage as well as renal cell progenitors were found in the placenta. The expression of MSC and renal progenitor surface markers varied throughout gestation, but was highest on E12-15 where such cells represented a small but significant percentage of the population. Of the studied TFs, 10 of 11 renal TFs were found at moderate to high levels, and all stem cell TFs were found. CONCLUSION: The mid-gestation murine placenta may serve as a source of multipotent stem cells and also contains cells which may be renal cell progenitors.

TBLR1 regulates the expression of nuclear hormone receptor co-repressors.

BACKGROUND: Transcription is regulated by a complex interaction of activators and repressors. The effectors of repression are large multimeric complexes which contain both the repressor proteins that bind to transcription factors and a number of co-repressors that actually mediate transcriptional silencing either by inhibiting the basal transcription machinery or by recruiting chromatin-modifying enzymes. RESULTS: TBLR1 [GenBank: NM024665] is a co-repressor of nuclear hormone transcription factors. A single highly conserved gene encodes a small family of protein molecules. Different isoforms are produced by differential exon utilization. Although the ORF of the predominant form contains only 1545 bp, the human gene occupies approximately 200 kb of genomic DNA on chromosome 3q and contains 16 exons. The genomic sequence overlaps with the putative DC42 [GenBank: NM030921] locus. The murine homologue is structurally similar and is also located on Chromosome 3. TBLR1 is closely related (79% homology at the mRNA level) to TBL1X and TBL1Y, which are located on Chromosomes X and Y. The expression of TBLR1 overlaps but is distinct from that of TBL1. An alternatively spliced form of TBLR1 has been demonstrated in human material and it too has an unique pattern of expression. TBLR1 and the homologous genes interact with proteins that regulate the nuclear hormone receptor family of transcription factors. In resting cells TBLR1 is primarily cytoplasmic but after perturbation the protein translocates to the nucleus. TBLR1 co-precipitates with SMRT, a co-repressor of nuclear hormone receptors, and co-precipitates in complexes immunoprecipitated by antiserum to HDAC3. Cells engineered to over express either TBLR1 or N- and C-terminal deletion variants, have elevated levels of endogenous N-CoR. Co-transfection of TBLR1 and SMRT results in increased expression of SMRT. This co-repressor undergoes ubiquitin-mediated degradation and we suggest that the stabilization of the co-repressors by TBLR1 occurs because of a novel mechanism that protects them from degradation. Transient over expression of TBLR1 produces growth arrest. CONCLUSION: TBLR1 is a multifunctional co-repressor of transcription. The structure of this family of molecules is highly conserved and closely related co-repressors have been found in all eukaryotic organisms. Regulation of co-repressor expression and the consequent alterations in transcriptional silencing play an important role in the regulation of differentiation.

The growth arrest-specific gene product Gas6 promotes the survival of human oligodendrocytes via a phosphatidylinositol 3-kinase-dependent pathway.

Microarray analysis revealed that transcripts for the Axl and Mer receptor tyrosine kinases are expressed at high levels in O4+-immunopanned oligodendrocytes isolated from second trimester human fetal spinal cord. In humans the sole known ligand for the Axl/Rse/Mer kinases is growth arrest-specific gene 6 (Gas6), which in the CNS is secreted by neurons and endothelial cells. We hypothesized that Gas6 is a survival factor for oligodendrocytes and receptor activation signals downstream to the phosphatidylinositol 3 (PI3)-kinase/Akt pathway to increase cell survival in the absence of cell proliferation. To test this hypothesis, we grew enriched human oligodendrocytes for 6 d on a monolayer of NIH3T3 cells stably expressing Gas6. CNP+ oligodendrocytes on Gas6-secreting 3T3 cells had more primary processes and arborizations than those plated solely on 3T3 cells. Also, a twofold increase in CNP+ and MBP+ oligodendrocytes was observed when they were plated on the Gas6-secreting cells. The effect was abolished in the presence of Axl-Fc but remained unchanged in the presence of the irrelevant receptor fusion molecule TrkA-Fc. A significant decrease in CNP+/TUNEL+ oligodendrocytes was observed when recombinant human Gas6 (rhGas6) was administered to oligodendrocytes plated on poly-L-lysine, supporting a role for Gas6 signaling in oligodendrocyte survival during a period of active myelination in human fetal spinal cord development. PI3-kinase inhibitors blocked the anti-apoptotic effect of rhGas6, whereas a MEK/ERK inhibitor had no effect. Thus Gas6 sustains human fetal oligodendrocyte viability by receptor activation and downstream signaling via the PI3-kinase/Akt pathway.

Hematopoiesis and stem cell renewal in long-term bone marrow cultures containing catalase.

Culturing mouse bone marrow in the presence of catalase dramatically alters hematopoiesis. Granulocyte output is initially increased 4- to 5-fold. This increase is transient and granulocyte production declines as immature (Sca-1+/LIN-) cells accumulate. One third of these immature cells have a phenotype (Sca-1+/c-Kit+) characteristic of hematopoietic stem cells. At 2 to 3 weeks there are greater than 200-fold more Sca-1+/c-Kit+/LIN- cells in treated cultures than in controls. This population contains functional stem cells with both short-term and long-term bone marrow repopulating activity. In addition to myeloid progenitors, this Sca-1+/LIN- population contains a large number of cells that express CD31 and CD34 and have an active Tie-2 promoter, indicating that they are in the endothelial lineage. After 3 to 4 weeks hematopoiesis in treated cultures wanes but if catalase is removed, hematopoiesis resumes. After 7 to 10 days the cultures are indistinguishable from untreated controls. Thus, protected from H2O2, hematopoietic progenitors multiply and become quiescent. This sequence resembles in vivo development in normal marrow. These results make it clear that peroxide-sensitive regulatory mechanisms play an important role in controlling hematopoiesis ex vivo and presumably in vivo as well. They also indicate that manipulation of the peroxide levels can be used to enhance the growth of hematopoietic stem cells in culture.